It is now recognized that lipid oxidation produces an array of compounds capable of initiating redox cell signaling. Some of the pathways induce apoptosis while others induce the synthesis of proteins which increase the threshold at which oxidative stress and cytotoxicity occurs. Understanding how these responses are distinguished is critical in determining the molecular events that protect the cell against oxidative damage mediated by xenobiotics or during the pathophysiology of disease. In the previous funding period the central focus of this proposal was to define the mechanisms through which oxidized lipids adapt the endothelial cell to oxidative stress using the induction of the intracellular antioxidant glutathione (GSH) as a model. Preliminary data and published observations developed through the previous funding period identified mechanisms through which a specific sub-class of oxidized lipids, those with electrophilic reactive carbon centers, mediate signal transduction. We found that cytoprotection was dependent on the activation of the electrophile response element (also known as the antioxidant response element) which induces the synthesis of cytoprotective proteins such as heme oxygenase and glutamyl cysteine ligase. In the course of these studies we have used a proteomics approach to define the subset of proteins reactive to electrophilic lipids in the cell which we have designated as the electrophile responsive proteome. These data have led to the hypothesis that electrophilic lipids generated during lipid peroxidation control cell function through modification of the proteins that compose the electrophilic responsive proteome. This hypothesis will be tested by pursuit of the following Specific Aims: 1) Determine whether distinct classes of lipid derived electrophilic cyclopentenones react with different members of the electrophile responsive proteome 2) Determine the effect of the activation of endogenous enzymatic sources of lipid oxidation products on the electrophile responsive proteome. 3) Determine the functional impact of the interaction of electrophilic lipids with endothelial cell mitochondria. The information gained from the accomplishment of these specific aims will give insight into the mechanisms of adaptation and cytotoxicity of the endothelium under toxicological and pathological stress.